Hello everyone,
I am currently using the H9-NSCs (Invitrogen, #N7800-100) and this is my first experience with neural stem cells and stem cells in general, so any help of any kind would be of a great value to me.
Few days ago I thawed a new stock of H9-NSCs (p4) on a PLO/Lamin-coated T25 flask. The first day the cell seemed fine there were no abnormalities of any kind (the top picture). However the next day (and gradually) I started to notice more and more floating cells and I noticed that the shape of cells is also changing and they seem to be dying for whatever reason (bottom picture). There are very very few cells that seem to be healthy but they are far from each other so i am not sure they can grow without the cell-cell contact?
I am very confused and I am not sure what I should do.. will it be useless to collect the floating cells and seed them again on a new flask?
If anyone had such issue before please let me know what did you and if there are any tips that i should know about the neural stem cells culture, I would be more than grateful to hear it out.
Thanks a lot
Hi Asma, getting cells back out of a frozen state can be challenging as their viability will depend on how well they were frozen in the first place and how stable the temperature they were kept at. Some amount of cell death is however expected and the dead cells will float in the liquid just as in your picture. This is not necessarily a huge concern. My advice would be to change the media daily to remove the dead floating cells. Please make sure you use fresh in date media components as this is very important. Also as a general note..when cells come out of the freezer it helps to seed them in a small dish. This depends on the cell count however you need to go for smaller dishes than you would with unthawed cells. So perhaps try 1 or 2 wells of a 6 well plate instead of going directly to T25. Hope this helps and good luck!
use ROCK inhibitor (10 microM) when plating NPCs. You need to comment on what coating you are using (vitronectin, laminin 510, gelating, polyornithin, or may be nothing?)
your bFGF stock may not be good. I assume you use fresh N2 and B27, and I assume you are using retinoic acid-free B27. I assume you are using Neurobasal or a similar medium. Your L-glutamine may be old, use glutaMax.
The stock may not be good. Though it is hard to start from scratch, try to read more about neural cultures, it is worthwhile to learn to understand and troubleshoot by yourself rather than relying on random advice. Here are our hESC-NPC papers, you can learn quite a lot by reading and following protocols. Invitrogen in general is not a great source to buy from (too big, hard to find a person who actually cultures). Try buying human NPCs from brainbits,com, and train yourself to culture NPCs. I am happy to help more if needed, https://pubmed.ncbi.nlm.nih.gov/16806174/, Article Long-Term, Stable Differentiation Of Human Embryonic Stem Ce...
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Veronica Biga Hi Veronica, thanks a lot for your very helpful tip~ it turns out the stock itself was the problem.. after several trials I finally have healthy cells I also have been using your suggestion (thawing in smaller dishes first then passing to T25) and it is working perfectly so far. Thx a lot for the help and all the best to you too ~
Igor O. Nasonkin Hi Igor, I have used ROCK inhibitor and the plates used were coated with PLo/Lamin. I used a freshly prepared N2/B27 (retinoic acid-free) and used glutamax. But the problem was not with the medium it was already with the stock itself (the conclusion after testing several stock vials and repeating the experiment with different coating and culture conditions).
I appreciate your kind advice and I totally agree with you that it is important to be able to troubleshoot your work by yourself and I am working on that. Thank you for sharing the papers with me I will check them all out~
Very kind of you indeed.
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